Described herein are methods for identifying and preparing high affinity nucleic acid ligands that bind human transforming growth factor xcex22 (TGFxcex22). The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential Enrichment. This invention includes high affinity nucleic acids of human TGFxcex22. Further disclosed are RNA ligands to TGFxcex22. Also included are oligonucleotides containing nucleotide derivatives modified at the 2xe2x80x2 position of the pyrimidines. Additionally disclosed are ligands to TGFxcex22 containing 2xe2x80x2-OCH3 purine modifications that may have higher stability in serum and in animals. This invention also includes high affinity nucleic acid inhibitors of TGFxcex22. The oligonucleotide ligands of the present invention are useful in any process in which binding to TGFxcex22 is required. This includes, but is not limited to, their use as pharmaceuticals, diagnostics, imaging agents, and immunohistochemical reagents.
Transforming growth factors betas (TGFxcex2s) are part of a superfamily of proteins that includes inhibins, activins, bone morphogenetic and osteogenic proteins, growth/differentiation factors, Mullerian-inhibiting substance, decapentaplegic and 60A (Drosophila), daf-7 and unc-129 (C. elegans), and vg1 (Xenopus) (Schlunegger and Grutter (1992) Nature 358:430-434). Three TGFxcex2 isotypes exist in mammals that are called TGFxcex21, TGFxcex22, and TGFxcex23. There is about 80% sequence identity between any pair of mammalian TGFxcex2s. TGFxcex2s bind to at least 5 receptors, but only 2 or 3 of them (types I, II, and possibly V) are signaling receptors. The intracellular signaling pathways activated by TGFxcex2s involve SMAD proteins and are being intensively studied (Padgett, et al. (1998) Pharmacol Ther 78:47-52). The signaling receptors are found on a variety of cells. In turn, a variety of cells express TGFxcex2s.
TGFxcex2s are synthesized as precursors composed of latency-associated protein (LAP) at the amino terminus and mature TGFxcex2 at the carboxyl terminus. The precursor is cleaved and assembles as a homodimer. TGFxcex2s are secreted from cells bound to LAP and latent TGFxcex2 binding proteins (LTBPs). Latent TGFxcex2s are released from LAP and LTBP and become active by a relatively uncharacterized mechanism that may involve proteolysis by plasmin or regulation by thrombospondin (Crawford, et al. (1998) Cell 93:1159-70). The mature, released TGFxcex2 homodimer has a combined molecular weight of xcx9c25000 daltons (112 amino acids per monomer). TGFxcex21 and TGFxcex22 bind heparin and there are indications that basic amino acids at position 26 are required for heparin binding (Lyon et al. (1997) Jour. Biol. Chem. 272:18000-18006).
The structure of TGFxcex22 has been determined using x-ray crystallography (Daopin, et al. (1992) Science 257:369-373; Schlunegger and Grutter (1992) Nature 358:430-434) and is very similar to the structure of TGFxcex21. TGFxcex2s belong to a structural family of proteins called the xe2x80x9ccysteine knotxe2x80x9d proteins that includes vascular endothelial growth factor, nerve growth factor, human chorionic gonadotropin, and platelet-derived growth factor. These proteins are structurally homologous, but have only 10-25% primary sequence homology.
The biological activities of the TGFxcex2s vary (Moses (1990) Growth Factors from Genes to Clinical Application 141-155, Wahl (1994) J. Exp. Med. 180:1587-1590). In some cases they inhibit cell proliferation (Robinson et al. (1991) Cancer Res. 113:6269-6274) and in other cases they stimulate it (Fynan and Reiss (1993) Crit. Rev. Oncogenesis 4:493-540). They regulate extracellular matrix formation and remodeling (Koli and Arteaga (1996) Jour. Mammary Gland Biol. and Neoplasia 1:373-380). They are also are very potent immunosuppressants (Letterio and Roberts (1998) Ann. Rev. Immunol. 16:137-161). TGFxcex2s are thought to play a significant role in fibrotic diseases, preventing the immune system from rejecting tumors (Fakhrai et al. (1996) Proc. Natl. Acad. USA 93:2090-2914), cancer cell growth (Koli and Arteaga (1996) J. Mammary Gland Bio. and Neoplasia 1:373-380, Reiss and Barcellos-Hoff (1997) Breast Cancer Res. and Treatment 45:81-85; Jennings and Pietenpol (1998) J. Neurooncol. 36:123-140), and tumor metastasis. They may have ancillary roles in autoimmune and infectious diseases. Inhibition of TGFxcex22 by an expressed antisense RNA (Fakhrai et al. (1996) Proc. Natl. Acad. USA 93:2090-2914) and by exogenous antisense oligonucleotides (Marzo et al. (1997) Cancer Research 57:3200-3207) has been reported to prevent glioma formation in rats.
The gene for mouse TGFxcex22 has been deleted (Sanford et al. (1997) Development 124:2659-2670). Mice lacking TGFxcex22 function die near birth and have aberrant epithelial-mesencymal interactions that lead to developmental defects in the heart, eye, ear, lung, limb, craniofacial area, spinal cord, and urogenital tracts. These defects, for the most part, do not overlap abnormalities that have been observed in TGFxcex21 and TGFxcex23 knockout mice. TGFxcex2s have also been overexpressed in cell lines or transgeneic mice (Koli and Arteaga (1996) J. Mammary Gland Bio. and Neoplasia 1:373-380, Bottinger et al. 1997 Kidney Int. 51:1355-1360; Bottinger and Kopp (1998) Miner Electrolyte Metab 24:154-160) with a variety of effects.
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichmentxe2x80x9d, now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,475,096, and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structurexe2x80x9d, now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA (See U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligandsxe2x80x9d, now abandoned, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeinexe2x80x9d, now abandoned, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed xe2x80x9cCounter-SELEXxe2x80x9d (See U.S. Pat. No. 5,580,737). U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEXxe2x80x9d, now abandoned, (See also U.S. Pat. No. 5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled xe2x80x9cFlow Cell SELEXxe2x80x9d, now U.S. Pat. No. 5,861,254, describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a Target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Revxe2x80x9d, now U.S. Pat. No. 5,496,938, describes methods for obtaining improved Nucleic Acid Ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEXxe2x80x9d, now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotidesxe2x80x9d, now abandoned, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2xe2x80x2-amino modifications (See U.S. Pat. No. 5,660,985). U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacementxe2x80x9d, now abandoned describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines. PCT/US98/00589, WO 98/18480 filed Jan. 7, 1998, entitled xe2x80x9cBioconjugation of Oligonucleotidesxe2x80x9d describes a method for identifying bioconjugates to a target comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5xe2x80x2-position of the nucleic acid ligands.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEXxe2x80x9d, now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEXxe2x80x9d, now U.S. Pat. No. 5,683,867 respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated herein by reference in their entirety.
The present invention includes methods of identifying and producing nucleic acid ligands to transforming growth factor beta (TGFxcex22) and the nucleic acid ligands so identified and produced. By substantially homologous it is meant a degree of amino acid sequence identity of 70% or more. In particular, RNA sequences are provided that are capable of binding specifically to TGFxcex22. Also included are oligonucleotides containing nucleotide derivatives modified at the 2xe2x80x2 position of the pyrimidines. Specifically included in the invention are the RNA ligand sequences shown in Tables 5, 7, 8, 11, 13, 14, 16-19, and FIG. 9 (SEQ ID NOS: 21-108 and 128-193). Also included in this invention are RNA ligands of TGFxcex22 that inhibit the function of TGFxcex22. Also described herein are 2xe2x80x2OMe-modified nucleic acid ligands of TGFxcex21.
Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to TGFxcex22, comprising the steps of (a) preparing a candidate mixture of nucleic acids, (b) contacting the candidate mixture of nucleic acids with TGFxcex22, (c) partitioning between members of said candidate mixture on the basis of affinity to TGFxcex22, and (d) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to TGFxcex22.
More specifically, the present invention includes the RNA ligands to TGFxcex22, identified according to the above-described method, including those ligands shown in Tables 5, 7, 8, 11, 13, 14, 16-19, and FIG. 9 (SEQ ID NOS: 21-108 and 128-193). Also included are nucleic acid ligands to TGFxcex22 that are substantially homologous to any of the given ligands and that have substantially the same ability to bind TGFxcex22 and inhibit the function of TGFxcex22. Further included in this invention are nucleic acid ligands to TGFxcex22 that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind TGFxcex22 and inhibit the function of TGFxcex22.
The present invention also includes other modified nucleotide sequences based on the nucleic acid ligands identified herein and mixtures of the same.